In this description, the following terms are employed:
Analyte - A substance or substances, either alone or in admixtures, whose presence is to be detected and, if desired, quantitated. The analyte may be a DNA or RNA molecule of small or high molecular weight, a molecular complex including those molecules, or a biological system containing nucleic acids, such as a virus, a cell, or a group of cells. Among the common analytes are nucleic acids (DNA and RNA) or segments thereof, either single- or double-stranded, viruses, bacteria, cells in culture, and the like. Also included are fungi, algae, other microorganisms, as well as animals (e.g., vertebrates) and plants, their cells, tissues and fluids.
Bridging Moiety - That moiety which on covalent attachment or non-covalent binding to the label of a polynucleotide sequence acts as a connection between the label and a signalling moiety.
Genomic DNA - An analyte comprising the DNA of an organism. Typically, the analyte will be purified nuclear DNA, and may, although not necessarily, include all nucleotide sequences present in the organism's DNA.
Label - That moiety attached to the probe by a linkage group which as is, or which after covalent attachment of a signalling moiety or a combination of bridging moiety and signalling moiety to it, or which after non-covalent binding of a signalling moiety or a combination of bridging moiety and signalling moiety to it, gives rise to a signal which is detectable, and in some cases, quantifiable.
Linkage Group - That moiety which serves to link or attach a label to the probe. The linkage group serves to hold the label away from the probe, so as to prevent interference with binding between the probe and target.
Probe - A polynucleotide sequence which is complementary to a target polynucleotide sequence in the analyte.
Signal - That characteristic of a label or signalling moiety that permits it to be detected.
Signalling Moiety - That moiety which on covalent attachment or non-covalent binding to the probe, label or to a bridging moiety attached or bound to the probe or label provides a signal for detection of the label and the moiety to which the label is attached.
Target - The specific sequence of bases in a nucleic acid present in an analyte whose presence is to be detected.
The analysis and detection of minute quantities of substances in biological and non-biological samples has become a routine practice in clinical and analytical laboratories. These detection techniques can be divided into two major classes: (1) those based on ligand-receptor interactions (e.g., immunoassay-based techniques) and (2) those based on nucleic acid hybridization (polynucleotide sequence-based techniques).
Immunoassay-based techniques are characterized by a sequence of steps comprising the non-covalent binding of an antibody and an antigen complementary to it. See, for example, T. Chard, An Introduction To Radioimmunoassay And Related Techniques (1978).
Polynucleotide sequence-based detection techniques are characterized by a sequence of steps comprising the non-covalent binding of a labelled polynucleotide sequence or probe to a complementary sequence of the analyte under conditions which permit hybridization of the bases through Watson-Crick pairing, and the detection of that hybridization. [M. Grundstein and D. S. Hogness, "Colony Hybridization: A Method For The Isolation Of Cloned DNAs That Contain A Specific Gene", Proc. Nat. Acad. Sci. USA, 72, pp. 3961-65 (1975); D. T. Kingsbury, "DNA Probes In The Diagnosis Of Genetic And Infectious Diseases", Trends In Biotechnology, 5, pp. 107-11 (1987).]
The non-covalent binding of a labelled sequence or probe to a complementary sequence of an analyte is the primary recognition event of polynucleotide sequence-based detection techniques. This binding event is brought about by a precise molecular alignment and interaction of complementary nucleotides of the probe and target. It is energetically favored by the release of non-covalent bonding free energy, e.g., hydrogen bonding, stacking free energy and the like.
In order to employ the non-covalent binding of a probe for the determination of an analyte containing a target sequence, it is necessary to be able to detect binding of the probe to the target. This detection is effected through a signalling step or event. A signalling step or event allows detection in some quantitative or qualitative manner of the occurrence of the primary recognition event.
In general, the use of a separate label moiety to attach a signal moiety to a probe is a desirable way to couple the primary recognition and signal events. First, this technique permits attachment of large bridging and signalling moieties to the probe, while interfering only minimally with the structure of the probe, and therefore with its binding to the target. Second, use of a separate label permits the indirect attachment of many signalling moieties to a single locus within a probe molecule. Attachment of a label bearing many signalling moieties at a single point, rather than attaching signalling moieties to many bases, serves to minimize the likelihood that binding between the probe and target will be disturbed, while providing a larger signal than with a single signalling moiety. An additional improvement is to employ a linkage group as a rigid point of attachment between the label and probe, which will hold the label away from the probe during hybridization.
The primary recognition event and the signalling event of polynucleotide sequence based detection techniques may be coupled either directly, proportionately or inverse proportionately. Thus, in such systems as nucleic acid hybridization assays performed with detectable probes, the amount of signal is usually directly proportional to the amount of analyte present. Inversely proportional techniques include, for example, competitive assays, wherein the amount of detected signal decreases with increasing amounts of analyte present in the sample.
Amplification techniques are of great importance when only a small amount of a target is present. For example, the signalling component of the assay may be present in a ratio of 10:1 for each recognition component, thereby providing a 10-fold increase in sensitivity.
A wide variety of signalling events may be employed to detect the occurrence of the primary recognition event. The signalling event chosen depends on the particular signal that characterizes signalling moiety employed. Although the label itself, without further treatment, may be detectable, more often, either the signalling moiety is attached covalently, or bound non-covalently to a label or a combination of signalling and bridging moieties in order to render the primary recognition event detectable.
Although the combination of bridging moiety and signalling moiety, described above, may be constructed before attachment or binding to the label, it may also be sequentially attached or bound to the label. For example, the bridging moiety may be first bound or attached to the label and then the signalling moiety combined with the joined label and bridging moiety. In addition, it should be understood that several bridging moieties and/or signalling moieties may be employed together in any one combination of bridging moiety and signalling moiety.
Examples of the covalent attachment of a signalling moiety or a combination of bridging moiety and signalling moiety to a label include chemical modification of the label with signalling moieties. In addition, the primary recognition event may be detected by the non-covalent binding of a signalling moiety or a combination of bridging moiety and signalling moiety that itself can be detected by appropriate means, or the non-covalent binding to the label of a combination of bridging moiety and signalling moiety to provide a signal that may be detected by one of those means. For example, the label could be bound to a bridging moiety, e.g., a lectin, and then bound through the lectin, or bridging moiety, to another moiety that is detectable by appropriate means.
There are a wide variety of signalling moieties and bridging moieties that may be employed for covalent attachment or non-covalent binding to the label of polynucleotide sequences useful as probes in analyte detection systems. All that is required is that the signalling moiety provide a signal that may be detected by appropriate means and that the bridging moiety, if any, be characterized by the ability to attach covalently or to bind non-covalently to the label, and also possess the ability to combine with a signalling moiety.
Signalling moieties may be radioactive or non-radioactive. Radioactive signalling moieties are characterized by one or more radioisotopes of phosphorous, iodine, hydrogen, carbon, cobalt, nickel, and the like. Preferably the radioisotope emits .beta. or .gamma. radiation, and has a long half life. Detection of radioactive signalling moieties is typically accomplished by the stimulation of photon emission from crystalline detectors caused by the radiation, or by the fogging of a photographic emulsion.
Non-radioactive signalling moieties have the advantage that their use does not pose the hazards associated with exposure to radiation, and that special disposal techniques after use are not required. [D. T. Kingsbury, (1987), p. 108.] In addition, they are generally more stable, and as a consequence, cheaper to use. Detection sensitivities of non-radioactive signalling moieties may be as high or higher than those of radioactive signalling moieties.
Among the preferred non-radioactive signalling moieties or combinations of bridging and signalling moieties useful with non-radioactive labels are those based on the biotin/avidin binding system [P.R. Langer et al., "Enzymatic Synthesis of Biotin Labeled Polynucleotides: Novel Nucleic Acid Affinity Probes", Proc. Nat. Acad. Sci. USA, 78, pp. 6633-37 (1981). R. H. Singer and D. C. Ward, "Actin Gene Expression Visualized In Chicken Muscle Tissue Culture By Using In Situ H-ybridization With A Biotinated Nucleotide Analog", Proc. Nat. Acad. Sci USA, 79, pp. 7331-35 (1982)]. For a review of non-radioactive signalling and bridging-signalling systems, see U.S. Pat. No. 4,711,955.
Non-radioactively labeled polynucleotides are not more widely used in detection systems because the attachment of a label which does not interfere with hybridization is expensive and because of difficulties in attaching the signalling moiety to the probe. The chemical reaction conditions that might be useful for modification of a polynucleotide to add it to a label are often too vigorous to be sufficiently selective for a particular nucleotide. More importantly, chemical labelling of polynucleotide sequences often interferes with the hydrogen bonding necessary for hybridization. For example, dicarbonyl reagents, such as glyoxal and kethoxal, react with guanine residues, but the glyoxal and kethoxal reacted nucleotides do not hybridize to complementary sequences in the analyte because the glyoxal or kethoxal moiety interferes with the hydrogen bonding necessary for hybridization [M. Litt, "Structural Studies on Transfer Ribonucleic Acid. I. Labeling Of Exposed Guanine Sites in Yeast Phenylalanine Transfer Ribonucleic Acid with Kethoxal", Biochemistry, 8, pp. 3249-53 (1969)].
An alternative approach, such as that disclosed in U.S. Pat. No. 4,711,955, provides for covalent attachment of a label to individual bases by way of a linkage group. Labeled probes must then be constructed from labeled and unlabeled bases using a polynucleotide complementary to the probe as a template. Thus little specificity as to the location of the label on the probe is possible, and as consequence, steric interference between adjacent labels is possible. In addition, synthesis of the labeled monomeric nucleotides prior to incorporation into the polynucleotides involves expensive chemical processes. The coupling of the labelled monomeric nucleotides into a polynucleotide is also expensive, as the cost of the enzymes used in enzymatic coupling is substantial.
A further deficiency of a labeling technique in which individual nucleotides of a probe sequence are labeled is that the signal intensity is generally low. Because a single signalling moiety is attached to each nucleotide base, in order to achieve a level of signal intensity that is readily detectable, a long probe molecule is required. This reduces the ability of the probe to detect minor mismatches between the probe and analyte, since sufficient hydrogen bonding between other bases will cause the probe to remain bound to the analyte during washing. Detection of single mismatches between base pairs is of critical importance in diagnosing certain inherited diseases such as sickle-cell anemia [B. J. Conner et al., "Detection of Sickle Cell .beta..sup.s -Globin Allele By Hybridization With Synthetic Oligonucleotides, Proc. Nat. Acad. Sci. USA, 80, pp. 278-82 (1983)].
A particularly important application of the present invention is in screening an organism's entire genome in order to detect a particular target base sequence. For example, it is desirable to detect the presence of inheritable diseases in potential human parents in cases where the disease does not manifest itself until after child bearing age has been attained, as in Huntington's chorea, and in diseases where heterozygous individuals are merely carriers, as in sickle cell anemia and Tay Sachs disease. Genomic screening requires a probe having great sensitivity. Moreover, effective genomic screening requires a short probe capable of detecting a mutation at a single base within the affected gene in order to detect inheritable diseases resulting from alteration at only a single base, such as sickle cell anemia Conner et al., 1983]. A probe capable of selectively detecting a target polynucleotide sequence in an analyte of genomic DNA or RNA may be termed a genomic probe. To date no genomic probes apart from those bearing radioactive signalling moieties and those greater than about 400 base pairs in length have been disclosed which are capable of distinguishing mutant from wild type individuals. As will be demonstrated below, short probes prepared according to the present invention are capable of doing so.